LAPTM4B counteracts ferroptosis via suppressing the ubiquitin-proteasome degradation of SLC7A11 in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide, necessitating the identification of novel therapeutic targets. Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is involved in biological processes critical to cancer progression, such as regulation of solute carrier transporter proteins and metabolic pathways, including mTORC1. However, the metabolic processes governed by LAPTM4B and its role in oncogenesis remain unknown. In this study, we conducted unbiased metabolomic screens to uncover the metabolic landscape regulated by LAPTM4B. We observed common metabolic changes in several knockout cell models suggesting of a role for LAPTM4B in suppressing ferroptosis. Through a series of cell-based assays and animal experiments, we demonstrate that LAPTM4B protects tumor cells from erastin-induced ferroptosis both in vitro and in vivo. Mechanistically, LAPTM4B suppresses ferroptosis by inhibiting NEDD4L/ZRANB1 mediated ubiquitination and subsequent proteasomal degradation of the cystine-glutamate antiporter SLC7A11. Furthermore, metabolomic profiling of cancer cells revealed that LAPTM4B knockout leads to a significant enrichment of ferroptosis and associated metabolic alterations. By integrating results from cellular assays, patient tissue samples, an animal model, and cancer databases, this study highlights the clinical relevance of the LAPTM4B-SLC7A11-ferroptosis signaling axis in NSCLC progression and identifies it as a potential target for the development of cancer therapeutics.

Membrane (Millipore, Cat#HATF00010).The membranes were blocked with 3% BSA in TBS containing 0.1% Tween-20 (TBST) for 1 hour at room temperature or 5% non-fat milk in TBST, and then incubated with primary antibodies overnight at 4°C.After four washes with TBST, the membranes were incubated with secondary antibodies for 1 hour at room temperature.Subsequently, the membranes were washed and incubated with Ultrasensitive ECL Chemiluminescent Substrate (Biosharp, Cat#BL523B), followed by imaging using ChemiCapture Imaging System (Clinx, Cat#6000Exp).The quantification of protein levels was performed by normalizing to an internal control protein using ImageJ software version 1.53C (NIH, Bethesda, MD; http://imagej.nih.gov/ij).

Data preprocessing and filtering for Metabolomics analysis
The raw mass spectrometry (MS) data underwent comprehensive processing using MS-DIAL software, encompassing peak alignment, retention time correction, and peak area extraction.Metabolite identification was achieved through the integration of accurate mass analysis (with a mass tolerance of less than 10 ppm) and MS/MS data comparison (with a mass tolerance of less than 0.02 Da) against established databases such as HMDB, massbank, and other publicly available repositories, as well as our own curated metabolite standard library.
To ensure data quality and reliability, only variables within the extracted-ion features that exhibited non-zero measurement values in at least one group, accounting for more than 50% of the total measurements, were retained for further analysis.This selection criterion aimed to focus on metabolites that exhibited consistent and meaningful variations across the experimental groups, ensuring robust statistical analysis and interpretation of the results.

Multivariate statistical analysis for Metabolomics analysis
All multivariate data analyses and modeling were conducted using R (version 4.0.3) and appropriate R packages, adhering to established academic practices.Prior to analysis, the data underwent mean-centering and Pareto scaling to account for variations in scale and to enhance comparability between variables.Several modeling techniques were employed in this study, including principal component analysis (PCA), orthogonal partial least squares for 12-14 days.Afterwards, the cells were fixed with methanol, stained with crystal violet, and subsequently imaged.

EdU assay
The current study utilized an EdU kit (Rib bio, China, Cat#C10310) to determine cellular proliferation.The cells in the growth phase were obtained at a density of 8 × 10 3 cells and were seeded into 96-well plates.Following treatment with 1-5 μM erastin for 24 hours, the EdU solution was diluted using cell complete medium at a ratio of 1000:1 to prepare an appropriate quantity of 50 μM EdU medium.Subsequently, 100 μL of the 50 μM EdU medium was added to each well and incubated for 2 hours.Afterward, each well was subjected to a 30 minutes incubation with 4% paraformaldehyde-containing PBS at room temperature to fix the cells.50 μL of 2 mg/mL glycine was added to each well, and the decolorizing shaker was incubated for 5 minutes before 100 μL of 0.5% TritonX-100 was added to each well and the decolorizing shaker was incubated for 10 minutes with 0.5% TritonX-100 in PBS.Then, 100 μL of 1X Apollo® staining solution was added and incubated for 30 minutes at room temperature on a decolorizing shaker, while being protected from light.Each well was washed by adding 100 μL of 0.5% TritonX-100 in PBS decolorizing shaker 2~3 times for 10 minutes each time.Furthermore, the DNA was stained with a 1X Hoechst33342 or DAPI solution (100:1) while being protected from light.The staining process was carried out by incubating for 30 minutes at room temperature on a decolorizing shaker.Finally, 100 μL of PBS was added, and inverted fluorescence microscope images were captured by the inverted fluorescence microscope (Zeiss, Axio Observer 3).

Measurement of cell apoptosis and PI assay
The Cell Apoptosis Kit (BestBio, Cat#BB-4101) was employed to measure the apoptosis, according to the instructions from manufacturer.After 24 hours of cell attachment, the cells were digested with EDTA-free trypsin and washed twice with pre-cooled PBS, then resuspended in 400 µL of 1x Annexin-V conjugate solution.5 µL of Annexin-V-FITC staining solution was added and the cells were incubated for 15 minutes at 2-8°C in the dark.
Subsequently, 5 µL of PI staining solution was added and the cells were again incubated for 5 minutes at 2-8°C in the dark.At least 10,000 cells were collected and analyzed using a flow cytometer (Beckman coulter, USA).
Western blot analysis of GPX4 protein levels in LAPTM4B-depleted A549 and H1299 cells.(B) Quantification of SLC7A11 mRNA expression in wild-type (WT) and LAPTM4B knockout (KO) cells measured by Q-PCR.Quantification of n=4 experiments, presented as mean ± SEM, data normalized to "A549".(C) Western blot analysis of SLC7A11 protein levels in A549 cells treated with 50 µg/mL cycloheximide (CHX) for the indicated times.Upper panel: Representative experiment.Lower panel: Quantification of n=3 experiments, presented as mean ± SEM.The red dashed line indicates the time point when half of the endogenous SLC7A11 has been degraded.(D) Western blot analysis of SLC7A11 protein levels in A549 cells treated with 1 µmol/L bafilomycin-A1 (BafA1) or 20 µmol/L MG-132, together with 50 µg/mL CHX for the indicated times.Upper panel: Representative experiment.Lower panel: Quantification of n=3 experiments, presented as mean ± SEM. * p<0.05.(E) Western blot analysis of LAPTM4B expression in LAPTM4B stably expressing cells compared to control cells, using anti-LAPTM4B and anti-Flag antibodies.(F) Measurement of the siRNAs' efficiency by Q-PCR in A549 cells.(G) WT and LAPTM4B KO H1299 cells were transfected with the indicated siRNA, and subsequent western blotting was performed to determine SLC7A11 protein levels.Upper panel: Representative experiment.Lower panel: Quantification of n=3 experiments, presented as mean ± SEM. * p<0.05.(H) WT and LAPTM4B KO H1299 cells were transfected with indicated siRNA.Immunoprecipitation of the cell lysate using SLC7A11 antibody, followed by immunoblotting with Ubiquitin antibody.Left panel: Representative experiment.Right panel: Quantification of at least three experiments, presented as mean ± SEM. * p<0.05.12 Supplementary Figure S6.The regulation of LAPTM4B on the SLC7A11 degradation is facilitated by NEDD4L/ZRANB1 (A) H1299 cells stably expressing LAPTM4B and control cells were treated with 50 µg/mL CHX for the indicated times, and SLC7A11 protein levels were assessed by Western blotting.Upper panel: Representative experiment.Lower panel: Quantification of n=3 experiments, mean ± SEM. * p<0.05.(B) Stably expressing LAPTM4B H1299 cells and the control cells were transfected with the indicated siRNA, and subsequent western blotting were performed to determine SLC7A11 protein levels.Upper panel: Representative experiment.Lower panel: Quantification of n=3 experiments, presented as mean ± SEM. * p<0.05.(C) Stably expressing LAPTM4B H1299 cells and the control cells were transfected with indicated siRNA.Immunoprecipitation of the cell lysate using SLC7A11 antibody, followed by immunoblotting with Ubiquitin antibody.Upper panel: Representative experiment.Lower panel: Quantification of at least three experiments, presented as mean ± SEM. * p<0.05.(D) LAPTM4B stably expressing H1299 cells and control cells were harvested to measure lipid peroxidation.Left panel: Representative experiment.Right panel: Quantification of n=3 experiments, mean ± SEM. p(H1299 Ctrl, H1299 LAPTM4B)=0.0031.(E)LAPTM4B overexpressing A549 cells and control cells were seeded at a density of 8 × 10 3 cells per well in 96-well plates.After treatment with 5 μM erastin for 24 hours, cells were stained with DAPI (blue) and EdU (red) to visualize proliferative cells.(F)LAPTM4B stably expressing H1299 cells (8 × 10 3 ) and control cells were seeded in 96well plates.After treatment with 5 μM erastin for 24 hours, cells were stained with DAPI (blue) and EdU (red) to visualize proliferative cells.Left panel: representative experiment.Right panel: quantification of n=3 experiments, mean ± SEM. p(H1299 Ctrl_DMSO, H1299 Ctrl_Erastin)=0.0187,p(H1299 Ctrl_DMSO, H1299 LAPTM4B_DMSO)=0.0342.p(A549 Ctrl_DMSO, A549 Ctrl_Erastin)=0.007,p(A549 Ctrl_DMSO, A549 LAPTM4B_DMSO)=0.0011,LAPTM4B stably expressing H1299 cells (4 × 10 3 ) and control cells were seeded in a 6well plate, treated with 5 μM erastin for 24 hours, and cultured at 37°C for 10 days.Cells were fixed with methanol, stained with crystal violet, imaged, and quantified.Left panel: representative experiment.Right panel: quantification of n=3 experiments, mean ± SEM. p(H1299 Ctrl_DMSO, H1299 Ctrl_Erastin)=1.378E-06,p(H1299 Ctrl_DMSO, H1299 LAPTM4B_DMSO)=0.006.(C) LAPTM4B stably expressing A549 cells were transfected with the indicated siRNA, and subsequent western blotting was performed to determine LAPTM4B and SLC7A11 protein levels.Right panel: quantification of n=3 experiments, mean ± SEM. p(A549 Ctrl_NC siRNA, A549 Ctrl_SLC7A11 siRNA)=0.0299,p(A549 Ctrl_NC siRNA, stably expressing H1299 cells were transfected with indicated siRNAs, and LAPTM4B and SLC7A11 levels were determined by Western blotting.(E) LAPTM4B stably expressing H1299 cells and control cells were transfected with SLC7A11 siRNA.72 hours after transfection, cells were harvested to measure oxidative ROS.(F) Quantification of ROS measurement results from (E).Quantification of n=3 experiments, mean ± SEM. p(H1299 Ctrl_NC siRNA, H1299 LAPTM4B_NC siRNA)=0.0403,p(H1299 LAPTM4B_NC siRNA, H1299 LAPTM4B_SLC7A11 siRNA)=0.0088.(G) LAPTM4B stably expressing A549 cells and control cells were transfected with SLC7A11 siRNA and seeded in 96-well plates.72 hours after transfection, cells were stained with DAPI (blue) and EdU (red) to visualize proliferative cells.PAGE gels and transferred onto LF-PVDF (Millipore, Cat#IPVH00010) or NC Transfer